The invention relates to virology and antiviral drug screening.
The development of anti-viral strategies against hepatitis C virus (HCV) infection has been hindered by the lack of an ideal animal model, or even a cell culture system, for HCV replication. One characteristic of an ideal HCV animal model or cell culture system would be the ability to induce expression of an infectious HCV in a cell in vitro or in vivo.
The HCV genome consists of a positive strand RNA that encodes a single precursor viral protein that is cleaved by cellular and viral proteases to generate viral structural and non-structural proteins, respectively. Non-translated regions 5xe2x80x2 and 3xe2x80x2 (5xe2x80x2NTR and 3xe2x80x2NTR) to the open reading frame encoding the precursor protein are also involved in viral replication. For a review of HCV, see Houghton, xe2x80x9cChapter 32, Hepatitis C Viruses,xe2x80x9d in: Fields Virology, 3rd ed., Fields et al. eds., pp 1035-1058, 1996, Lippincott-Raven Publishers, Philadelphia, Pa.
Infection with HCV is one of the leading causes of chronic liver disease throughout the world. Chronic infection nearly always ensues after acute exposure to HCV, and chronically infected individuals develop cirrhosis and hepatocellular carcinoma at a dramatically elevated rate compared with the rate of disease in an uninfected population.
The development of more effective treatments has been limited by the lack of an effective tissue culture or small animal model of infection. HCV replication systems based on a self-replicating HCV RNA replicon is dependent only on expression of the nonstructural viral proteins. Therefore, such systems do not recapitulate all steps of the HCV virus life cycle, some of which may be effective targets for antiviral intervention. Cell lines stably transfected with the HCV precursor are not capable of HCV RNA replication. Consequently, such cell lines cannot be used to screen for antiviral drugs that block viral RNA replication. RNA transcripts from an infectious HCV cDNA clone can replicate in chimpanzees, but any model that requires the use of large, expensive primates is impractical.
To address the historical and intractable limitations in the above HCV replication models, the invention provides an inducible system for producing infectious or replicative HCV, as described in the Example below, thereby fulfilling a long-felt need for an ideal HCV replication system.
The invention is based on the development and implementation of an inducible cell-based system for producing replicative HCV. Because the HCV produced by this system is replicative and has the full complement of genetic material found in HCV (e.g., naturally occurring HCV), all aspects of the HCV life cycle can be examined, e.g., in screening assays for candidate antiviral compounds. The inducibility of the system allows the propagation of cells or animals containing HCV genetic material without the damaging effects of HCV replication. Thus, viral replication is induced only when desired, for example, in a particular step that requires infectious HCV replication in an assay.
Accordingly, the invention features a nucleic acid having a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme (e.g., a hepatitis D virus ribozyme), an inducible promoter (e.g., a T7 promoter) operably linked to the first and second nucleotide sequences, and optionally a transcription termination signal (e.g., a T7 transcription termination signal) operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3xe2x80x2 sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible promoter and optionally terminated by the transcription termination signal. The invention also includes cells that harbor a nucleic acid of the invention.
A method of producing infectious HCV is also included in the invention. A cell containing the nucleic acid of the invention (e.g., as an episome or an integrated cassette) can then be used to generate infectious HCV by inducing the inducible promoter of the nucleic acid. For example, if the promoter is a T7 bacteriophage promoter, HCV is produced by expressing a T7 RNA polymerase in the cell. The T7 RNA polymerase in turn can be expressed by infecting the cell with a viral vector (e.g., a vaccinia vector) encoding the T7 RNA polymerase. Alternatively, the cell can contain an episomal plasmid or genomic transgene (e.g., delivered by a retrovirus) that expresses T7 RNA polymerase. Regardless of the vectors used to express T7 RNA polymerase, the expression of the polymerase can itself be regulated, depending on the genetic elements operably linked to the sequence encoding the polymerase.
The invention further includes a screening method for identifying a compound (e.g., a polypeptide, small molecule, or nucleic acid, such as an antisense nucleic acid or ribozyme) that inhibits replication of an HCV. The method includes (1) providing a test cell containing a nucleic acid of the invention, (2) inducing the inducible promoter of the nucleic acid, (3) contacting the test cell with a candidate compound, and (3) detecting a decrease in the amount of infectious hepatitis C virus produced by the test cell compared to the amount of the infectious hepatitis C virus produced by a control cell. The detecting step can include measuring (e.g., by PCR) the amount of negative strand hepatitis C viral RNA in the cell or the amount of positive strand hepatitis C viral RNA in the cell or in cell-free virions produced by the cell.
Since it is possible that the candidate compound inhibits HCV replication by inhibiting a viral or cellular protease responsible for cleaving the HCV precursor protein, the screening method can further include determining whether a hepatitis C virus structural or non-structural protein is cleaved from a hepatitis C virus precursor protein in the cell after the contacting step, e.g., by protein gel electrophoresis.
As used herein, xe2x80x9cinhibitsxe2x80x9d or xe2x80x9cinhibitionxe2x80x9d means any measurable decrease (e.g., 10%, 20%, 50%, 90%, or 100%) in an activity of interest.
As used herein, an xe2x80x9cinfectious hepatitis C virusxe2x80x9d means an HCV that is capable of propagation in a population of cells in vivo or in vitro. Therefore, an infectious hepatitis C virus minimally contains (1) a sequence encoding a precursor protein and (2) 5xe2x80x2 and 3xe2x80x2 non-translated flanking sequences sufficient to support virus replication (i.e., each step of the virus life cycle) in a cell population.
By one genetic element being xe2x80x9coperably linkedxe2x80x9d to another is meant that a genetic element (either in a plus strand, minus strand, or double stranded form) is structurally configured to operate or affect another genetic element. For example, a promoter operably linked to a sequence encoding a polypeptide means that the promoter initiates transcription of a nucleic acid encoding the polypeptide, and a transcription termination signal operably linked to the sequence encoding the polypeptide means that the transcription termination signal terminates transcription of a nucleic acid encoding the polypeptide.
The nucleic acids and methods of the invention provide a HCV replication system amenable to comprehensive, yet relatively inexpensive (as compared to infection of a primate), antiviral drug screening methods. Because the HCV replication system performs all steps of the virus life cycle, candidate antiviral compounds can be screened for activity against any vital viral or cellular drug target involved in virus replication. In addition, the HCV replication system is inducible, thereby allowing cells to vigorously replicate in the absence of HCV until virus replication becomes necessary for the particular step of a screening assay performed. Thus, the nucleic acids and methods of the invention remove a substantial obstacle in anti-HCV drug development.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.